1. Field of the Invention
The present invention relates to systems and methods for controlling the position of one or more slugs, droplets, plugs, boluses, etc. (hereinafter “slugs”). More particularly, aspects of the present invention relate to systems and methods for identifying and controlling the position of a slug in a microfluidic device.
2. Description of the Background
Devices for performing chemical, biological, or other reactions often feature one or more microfluidic channels having thermal control elements that are used to subject one or more reactants to a desired thermal profile. An example of such a device includes a microfluidic device for performing polymerase chain reaction (PCR) amplification of DNA molecules, or a microfluidic molecular diagnostic platform that performs PCR on a patient sample and then uses the PCR product for genotyping by performing a high resolution melt analysis. A description of PCR amplification, and an example of one possible microfluidic device including thermal control elements for PCR amplification and thermal melt analysis, are provided in U.S. patent application Ser. No. 12/165,043, which is hereby incorporated by reference.
In many applications of such devices, such as PCR amplification and thermal melt analysis, one or more fluorescent dyes are used to indicate various states of the reactants. For example, dyes such as the Alexa Fluor family of fluorescent dyes produced by Molecular Probes, the LCGreen dyes produced by Idaho Technology Inc., or SYBR Green can be used to monitor the temperature of reactants as well as other chemical properties.
Accurately controlling and monitoring the reactions, for example, by accurately placing the slugs of reactants in proximity to the thermal control elements and accurately reading the fluorescence of the slugs, may require determining the location of each slug, the boundaries between adjacent slugs, or other features of slugs within a channel. One method of facilitating the identification of individual sample slugs (i.e., slugs containing reactants) is to use a spacing or blanking slug with an alternative dye in between each sample slug. For example, the spacing or blanking slug may contain a first color dye (such as a red dye, e.g., Alexa Red or Alexa Fluor 647) while the samples contain an intercalating dye of a second color (such as an intercalating green dye, e.g., LCGreen or Sybr green). However, many dyes are known to have temperature dependent fluorescence. In some instances, the fluorescence of these dyes can change by a factor of two or more over the range of temperatures typically used for PCR amplification and thermal melt analysis. The dyes generally tend to lose their fluorescence intensity when heated, and gain fluorescent intensity when cooled.
Because the fluorescence of the dyes will increase when the slugs are cooled and decrease when the slugs are heated, slugs including temperature-dependent dyes may appear to move (for example, expanding and contracting) during thermal cycling. If real-time feedback (such as an imaging system using a signal intensity threshold approach) is desired for control of the flow of slugs through a microfluidic device, this apparent movement may interfere with a system's ability to perform fluid control (e.g., accurately placing the slugs in thermal contact with the thermal control elements) and to analyze the slugs.
Accordingly, what is desired are systems and methods for accurately and precisely controlling slugs during thermal cycling.